1a.Objectives (from AD-416):
The overall objective of the proposed research is to develop improved plant germplasm and advanced process technology enabling domestic production of two strategic industrial raw materials – domestic natural rubber and castor oil, along with their valuable co-products. The crops for industrial production of rubber are Parthenium argentatum (guayule), Taraxacum kok-saghyz (Russian dandelion) and Hevea brasiliensis (Hevea), and for castor oil are Ricinus communis (castor) and Lesquerella fendleri (Lesquerella).

Domestic production of both castor and rubber has taken place in the past, yet critical issues denied sustainability of these important industrial crops. We propose to address these via the following objectives:

Objective 1: Develop germplasm that enables domestic commercial production of natural rubber and ricinoleate by metabolic engineering and conventional breeding.

Objective 2: Develop technologies to enable the domestic production of natural rubber and ricinoleate with properties as good as or better than existing sources for industrial applications. Identify chemical processes and/or modifications to develop superior performance properties of natural rubber from domestic sources. Identify processing methods to enhance the value of castor oil byproducts.

1b.Approach (from AD-416):
Improvements in natural rubber yield will be sought through genetic manipulation of guayule and Russian dandelion plants. The biosynthesis of ricinoleate in Lesquerella will be metabolically engineered. Transformation methods will be developed and optimized; efficacious high expression and tissue-specific promoters will be identified. Genetically altered lines will be phenotypically characterized for alterations in enzymatic activity and production of target metabolites. Promising lines will be tested in controlled environment greenhouse and/or field trials on site or at collaborator locations. Improved germplasm will also be developed/identified through characterization of NPGS castor and Russian dandelion lines. Mutagenesis will be used to develop high yield, low toxin and allergen castor lines.

More specific knowledge of the biosynthetic pathways for production of natural rubber and hydroxy fatty acids will be achieved through physiological, cell biology, and biochemical studies.

Knowledge of the detailed physical and chemical characteristics of natural rubber from alternative crops will be developed to elucidate the mechanisms for differences from incumbent materials and to provide strategies for technological equivalence.

The processing of castor seed meal by bioconversion to reduce toxin and allergen content of byproducts will be researched.

3.Progress Report:
Progress was made on all project objectives under National Program 306. Under Obj 1: Develop germplasm that enables domestic commercial production of natural rubber and ricinoleate by metabolic engineering and conventional breeding, we have cloned a napin promoter from Rapeseed and created transgenic Lesquerella lines confirming the promoter confers seed-specific expression. The napin promoter is useful in engineering high hydroxyl fatty acid levels in Lesquerella. Constructs were developed to silence two fatty acid pathway genes to increase production of hydroxyl fatty acids of specific structure. Chloroplast transformation of guayule was successful, for the first time providing a new method to insert important rubber pathway genes. Chloroplast transformation allows high levels of foreign proteins to be produced while preventing escape of foreign genes, since guayule pollen contains no chloroplasts. Seed from the Kazakhstan Russian dandelion collection (NPGS, Pullman, Washington) was evaluated for its amenability in vitro shoot regeneration. Three confirmed diploid lines with good shoot regeneration were identified for genetic transformation. Computational analysis of Russian dandelion expressed sequences revealed that a key enzyme (HMGR) which regulates production of the monomer (IPP) of rubber polymer is a member of a gene family. Organ specific and temporal/developmental expression analysis of all the HMGR family members are in progress. Finally, field evaluations of castor confirmed the viability of recurrent selection to develop castor cultivars with increased oil yield. Under Obj 2: Develop technologies to enable the domestic production of natural rubber and ricinoleate with properties as good as or better than existing sources for industrial applications, new molecular species of diacylglycerols, triacylglycerols and tetraacylglycerols in the seed oil of L. fendleri were identified by original analytical methods. Fatty acids containing hydroxyl groups and double bonds were also identified. These biobased chemicals resemble those found in castor and may provide alternate materials for industry. The toxin ricin continues to be a concern as a byproduct of castor oil production, both as a threat agent and as a biohazard. A promising approach for eliminating ricin during castor oil production was developed, by proteolysis during organic solvent extraction. Initial attempts at bioconversion of seed meal remaining after oil has been extracted from castor seed yielded a product with potential use in hydrogels and a other non-food uses. A recent concern raised in some states is the presence of volunteer castor plants in fields resulting in contamination of the field crop with castor seeds. We have demonstrated that low level herbicide (glyphosate) spraying may be effective in eliminating castor volunteers from most fields of major crops. A solvent extraction process for guayule natural rubber was optimized to successfully recover solid guayule natural rubber over multiple harvests/extraction runs. Finally a quick method for detection of guayule resin in rubber, using a UV-detector during molecular weight measurements, was demonstrated.

4.Accomplishments
1.
Chloroplast transformation of guayule. Guayule is a native crop under commercial development in the South West of the United States of America for production of natural rubber and bioenergy. Increasing the yield of natural rubber, using the tools of biotechnology, could significantly impact the economic sustainability of this new crop. Agricultural Research Service scientists in Albany, California, developed a successful method for chloroplast transformation of guayule, for the first time providing a new method to insert important rubber pathway genes. Chloroplast transformation is especially attractive in that it allows high levels of foreign proteins to be produced while preventing escape of foreign genes, since guayule pollen contains no chloroplasts. Genetically-modified guayule with high levels of natural rubber could be developed with this technology.

2.
A Napin promoter directs seed specific expression in Lesquerella fendleri. To engineer seed oils or fatty acid contents in Lesquerella, a promising industrial oil seed for the arid west, it is preferable to utilize seed specific promoters, to limit the changes of oils and fatty acids to the seed, and to avoid the changes in membrane lipids in other parts of the plant. Agricultural Research Services scientists in Albany, California, have cloned and sequenced the upstream regulatory region of napA gene from Rapeseed (Brassica napus) and tested the tissue specific expression pattern of this promoter in Lesquerella. Five independent transgenic lines were grown to maturity and generated transgenic seeds. The activity of napA promoter was examined in various organs and tissues, including leaf, stem, root, flower and developing seeds. For eleven transgenic lines, the napA promoter showed activity only in developing seeds, not in any other organs and tissues. The napA promoter can be used to express a target gene and to facilitate future research on genetic engineering of seed oil in Lesquerella.

3.
Reducing ricin as a hazard of castor oil production. The toxin ricin continues to be a concern as a byproduct of castor oil production, both as a threat agent and as a biohazard. Researchers at the Western Regional Research Center in Albany, California, have identified an approach that improves processing of the castor seed, treating it with an enzyme that eliminates the ricin protein from the seed cake, making it safe to handle. A recent concern raised in some states is the presence of volunteer castor plants in fields resulting in contamination of the field crop with castor seeds if castor becomes a widely cultivated crop. We have demonstrated low level herbicide (glyphosate) spraying, may be effective in eliminating castor volunteers from most fields of major crops. In cooperative research with another research unit at the Western Regional Research Center and a collaborator in Spain, we have identified castor cultivars with reduced levels of ricin for inclusion in breeding programs to eliminate ricin from castor. These approaches can help to eliminate ricin as a problem and a perceived concern, thus fostering domestic production of castor oil, a key chemical feedstock with yields of up to 2500 lbs. of oil/acre, equivalent to replacing 8 barrels of petroleum with a renewable product that can be converted to uses currently supplied by petroleum.